PMID: 19352168 DOI: 10.1097/ALN.0b013e31819daedd
Materials and Methods
The study protocol was approved by the Home Office (London, United Kingdom) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986.

In Vitro  Experiments
Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups (Harlan Laboratories, Huntingdon, United Kingdom) and cultured by the interface method21,22with some modifications. In brief, the brain was quickly dissected and placed in ice-cooled (4°C) dissection solution. All stages of slice preparation were performed under sterile and ice-cooled conditions. Excess tissue (including the cerebellum, olfactory bulbs, and meninges) was removed, and the brain was cut into 400-μm sagittal slices using a McIllwain Tissue Chopper (Mickle Laboratory, Cambridge, United Kingdom). Under a dissecting microscope and avoiding contact with the hippocampus, the slices were separated using fine forceps. Slices containing the intact hippocampus were selected and positioned onto 30-mm-diameter semiporous cell culture inserts (five slices per insert) (Falcon; Becton Dickinson Labware, Millipore, Bedford, MA) and placed in a six-well tissue culture tray (Multiwell; Falcon, Becton Dickinson Labware). Eagle minimum essential medium enhanced with heat-inactivated horse serum (1.5 ml) was then transferred to each well.

The slices were incubated for 24 h in humidified air at 37°C, enriched with 5% carbon dioxide. The culture medium was replaced the next day with fresh, temperature-equilibrated medium before exposure to gas treatments. The groups of slices (n = 15 per group) were assigned to control (air + 5% carbon dioxide), dexmedetomidine 1 μm, gabazine 50 μm, 0.75% isoflurane, 0.75% isoflurane + dexmedetomidine 1 μm, and 0.75% isoflurane + gabazine 50 μm.

All subsequent gas exposure occurred in a specially constructed exposure chamber as previously described.23The gases, warmed by a water bath, were delivered in the headspace above the slices by a standard anesthetic machine at 2–3 l/min, and concentrations were monitored with an S/5 spirometry module (Datex-Ohmeda, Bradford, United Kingdom). After 3–4 min of gas flow, the chambers were sealed and placed in a 37°C incubator for 6 h (Galaxy R Carbon Dioxide Chamber; Wolf Laboratories, Pocklington, York, United Kingdom). After exposure, the slices were returned to the incubator for a further 12 h of culture to allow for suitable caspase-3 expression and then fixed overnight in 4% paraformaldehyde and subsequently immersed in 30% sucrose for a further 24 h at 4°C before slicing with a cryostat.

In Vivo  Experiments
Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n = 6 per group). Three doses of saline or dexmedetomidine (1, 10, or 25 μg/kg) were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). One group received 0.75% isoflurane, 25 μg/kg dexmedetomidine, and 500 μg/kg nonselective α2adrenoceptor antagonist atipamezole in 3 doses over the 6-h exposure (n = 4 per group). An additional three doses of 75 μg/kg dexmedetomidine in air were given to establish at extreme doses of dexmedetomidine whether apoptosis could be induced (n = 6 per group).

The animals were sacrificed (with 100 mg/kg sodium pentobarbital by intraperitoneal injection) at the end of gas exposure and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1 m buffer. After removal of the brain and storage overnight at 4°C in paraformaldehyde, it was transferred to 30% sucrose solution with phosphate buffer and 1% sodium azide and kept at 4°C until the brains were sectioned and stained immunohistochemically for caspase-3.

Immunohistochemistry
For the in vitro  experiments, the slices were sectioned at 25-μm intervals using a cryostat, and the inner sections were mounted onto Super Plus-coated glass slides (VWR International, Lutterworth, United Kingdom). The sections were allowed to dry at 37°C for 24 h and then immunostained while adherent to the slides. Concerning the in vivo  experiments, the brain was sliced at 30-μm intervals beginning at −3.6 mm from the bregma, the sections were then transferred to a six-well plate containing phosphate-buffered saline. Sections were dried at 37°C for 24 h and then immunostained while adherent to the slides, before preincubation with hydrogen 0.3% peroxidase in methanol for 30 min and then rinsed in phosphate-buffered saline. The sections were then incubated overnight at 4°C with rabbit anti-cleaved caspase-3 (1:2,500; New England Biolab, Hitchin, United Kingdom) and then washed three times in phosphate-buffered saline with 3% Triton at room temperature. Biotinylated secondary antibodies (1:200; Sigma, St. Louis, MO) and the avidin-biotin-peroxidase complex (Vector Laboratories, Orton Southgate, Peterborough, United Kingdom) were applied. The sections were again washed in phosphate-buffered saline before incubating with 0.02% 3,3′-diaminobenzidine with nickel ammonium sulfate in 0.003% hydrogen peroxide (DAB kit, Vector Laboratories). The sections were dehydrated through a gradient of ethanol solutions (70–100%) and then mounted (floating section) and covered with a cover slip.

Neurocognitive Evaluation
Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n = 6 per group). Three doses of saline or 25 μg/kg dexmedetomidine were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). The animals were allowed to mature until postnatal day 40 and then tested for hippocampal-dependent memory and learning function in a previously reported contextual fear-conditioning behavioral paradigm24in which the rats were taken from the vivarium in the behavioral room on the first test day and allowed to sit undisturbed in their homecage for 10 min. Once placed in the conditioning chamber, the rats were allowed 198 s of exploration.

The conditioning chamber was cubic (30 cm × 24 cm × 21 cm; Med Associates, Inc., St. Albans, VT) and had a white opaque back wall, aluminum sidewalls, and a clear polycarbonate front door. The conditioning box had a removable grid floor and waste pan. Between each rat, the box was cleaned with an almond-scented solution and dried thoroughly. The grid floor contained 36 stainless steel rods (diameter, 3 mm) spaced 8 mm center to center. When placed in the chamber, the grid floor made contact with a circuit board through which a scrambled shock was delivered. During training and context testing, a standard high efficiency particulate air filter (HEPA) filter provided background white noise of 65 db.

Afterwards, all animals received 6 cycles of 214 s of trace fear conditioning. The tone was presented for 16 s (2 kHz) followed by a trace interval of 18 s and subsequent foot shock (2 s, 0.85 mA). The rats were removed from the conditioning chamber 198 s after the last shock and returned to their home cage. The total time of the acquisition phase was 26 min. Acquisition time was defined as the time spent immobile after a shock divided by the intertrial interval. On the next day, trained rats were exposed to the same acquisition environment but received neither tone nor shock for 8 min (context test). The percentage of time an animal froze during the 8-min observation periods was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min (i.e. , 60 observations). Freezing time was assessed using VideoFreeze software (Med Associates Inc., Burlington, VT); therefore, the assessment can be considered objective. The percentage of freezing time (context results) and the area under curve were derived from plots between the percentage freezing time and trial time in the tone test and were used for statistical comparison (mean ± SD, n = 6 per group).

Statistical Analyses
The number of caspase-3–positive neurons in the cortex, thalamus, and hippocampus in each brain slice were counted by an observer blinded to the experimental protocol. Four brain slices were counted per animal. The immunohistochemical and behavioral data are presented as mean ± SD. Statistical analyses was performed by ANOVA followed by post hoc  Newman Keuls testing using the INSTAT (London, United Kingdom) program. P < 0.05 was set as significant.